A comparative study of mPCR, MLPA, and muscle biopsy results in a cohort of children with Duchenne muscular dystrophy: A first study
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.152635
Source of Support: None, Conflict of Interest: None
Background: Multiplex ligation-dependant probe amplification (MLPA) is a highly sensitive and rapid alternative to multiplex polymerase chain reaction (PCR). Muscle biopsy should be reserved for mutation-negative cases.
Keywords: Duchenne muscular dystrophy; immunohistochemistry; multiplex polymerase chain reaction (mPCR); multiplex ligation dependant probe amplification; muscle biopsy
Duchenne muscular dystrophy (DMD) is a progressive X-linked recessive neuromuscular disorder caused by mutations in the dystrophin gene located at Xp 21 chromosome.  With an incidence of 1 in 3500−5000 live born males, DMD is the most common lethal muscle disorder in children.  Dystrophin gene is the largest known human gene consisting of 79 exons, which encodes a 14kb mRNA. , Although immunohistochemistry/western (lc w) blotting remains the gold standard for diagnosing DMD  , lack of any effective treatment has emphasized the need for prenatal diagnosis and carrier detection. With biopsy, one can only make a definitive diagnosis, but this does not help in genetic counseling. Multiplex PCR technique as described by Chamberlain et al. and Beggs et al. has offered a rapid and less-invasive screening tool for detecting deletions in the central and 5' end hot spot regions of the dystrophin gene. , Deletions account for about two-thirds of the mutations in dystrophin gene and mPCR allows detection of 98% of those deletions. ,, This technique detects large deletions in about 60−65% patients, has largely replaced biopsy, and has become the preferred method of diagnosis in many developing countries like India.  However, it is qualitative and does not detect duplications, which account for 6% of mutations in DMD gene. ,, Multiplex ligation-dependant probe amplification (MLPA), originally developed by Schouten et al., offers a reliable quantitative method to detect deletions and duplications in all 79 exons of the dystrophin gene and also carrier testing. , MLPA adds another 10−15% positive cases to mPCR. Many neurologists, particularly in India, still perform muscle biopsies to diagnose children with DMD, and this should be performed only after available genetic testing is negative for the mutation.
In this prospective study, we describe the comparative mutational findings by both mPCR and MLPA testing in 83 suspected children of DMD followed by muscle biopsy to confirm the diagnosis of DMD in mutation-negative cases. It is important to consider more advanced genetic testing such as next-generation sequencing (NGS) to diagnose the MLPA-negative cases and, thus, further reduce/avoid the invasive muscle biopsy procedure, which does not assist in genetic counseling.
Ethical approval for this study was obtained from the institute ethics committee. Written informed consent from the parents/guardian and assent forms were obtained before recruiting the children for the study. This is a prospective study conducted over a 12-month period during 2012−2013, wherein 83 non-random, clinically suspected cases of DMD were recruited from the neurology services/neuromuscular disorders clinic for initial genetic analysis. The collected blood samples of 3 ml each were sent for genetic analysis first by mPCR technique followed by MLPA. Comparative analysis was done to determine the sensitivity and mutational pattern in the two genetic testing methods and also muscle biopsy in MLPA-negative cases as tools for diagnosing DMD.
Multiplex PCR: Genomic DNA was isolated from peripheral blood leukocytes and the test was performed as described by Chamberlain et al. and Beggs et al. for 30 exons corresponding to the hot spot regions. ,
Multiplex ligand-dependant probe amplification (MLPA): The MLPA reaction was performed to screen exons of the dystrophin gene using the SALSA MLPA probe sets P034 and P035 (MRC-Holland, Amsterdam, the Netherlands), according to the manufacturer's instructions. The MLPA samples consisted of approximately 200 ng of genomic DNA. Ligation and amplification were carried out on an ABI 9600 Thermal Cycler. All amplified fragments were separated using capillary electrophoresis on an ABI PRISM 3130 Genetic Analyzer.
Open method was used for obtaining the samples from either the biceps or quadriceps muscle. Immunohistochemical staining was carried out on fresh frozen sections to monoclonal antibodies against dystrophin (1, 2, 3), sarcoglycans (α,β,δ,γ), merosin (α2 laminin) as primary, and HRP tagged NOVO-linked secondary antibody (Novocastra Laboratories, Newcastle, UK).
Descriptive results were expressed as mean and standard, standard deviation for continuous variables and as the frequency (percentage) for categorical variables. Non-parametric Spearman correlation coefficients were calculated to study the correlation between parameters. All tests were two-sided, and the level of significance was fixed at 0.05.
The mean age of onset was 45.3 ± 25.2 months. The mean duration of illness was 53.3 ± 30.8 months. Majority of the children had delayed acquisition of motor milestones. About 11.9% patients had a history of delayed mental milestones. The predominant symptoms were difficulty in rising from the floor or low chair, climbing stairs and frequent falls. There was a history of consanguineous marriage in 18%. Similar family history was elicited in 32.0% of patients. On physical examination, calf hypertrophy was noted in 94.1% of patients. Other hypertrophied muscles were brachioradialis (9.9%), extensor digitorum brevis (EDB] (10.9%), deltoid (8.9%), quadriceps (4%), and tongue (10.4%). Toe walking was noted in 31.2% and weakness of the pectoral girdle in 25.2% of patients. Winging of the scapula was observed in 85(42.1%) patients. Contractures were common and present at tendoachilles in 75.7%, iliopsoas in 14.9%, hamstrings in 17.3%, and biceps in 4%. The mean creatinine kinase (CK) value was 12136.1 ± 8591.1 LU/L.
Of the 83 samples tested, mPCR detected deletions in 60 cases accounting for 72.3% positivity. Among these 60 deletions, 8 (8.6%) were proximal, 51 (54.8%) were distal, and 1 showed proximal and distal deletions [Figure 1]. While majority of the mutations involved less than 5 exons [34 (36.6%)], mPCR could identify 2 large deletions (2.2%) involving greater than 10 exons [Figure 2]. Deletion in the hot spot region of 44−55 exons was found in 50 (83.3%) cases. There were 22 single exon deletions (36.6%) in total. Most common deletions were of exon 45 in 10 cases (16.6%) followed by exon 51 and 45 - 52 exons in 6 cases each (10%).
All the 83 samples were further subjected to MLPA analysis, which detected deletions in an additional six cases and duplications in six (6.5%) mPCR-negative samples, thus increasing the combined detection rate of deletions to 79.5%. The rate of duplication detection was 7.2% for the whole cohort. MLPA detected 14 (15.1%) proximal and 57 (61.3%) distal deletions while, in one case, both proximal and distal deletions were found [Figure 1]. As with mPCR, the majority of mutations involved less than 5 exons [41 (44.1%)], but MLPA was able to detect 6 (6.5%) large mutations that involved more than 10 exons [Figure 2]. About 54 mutations were picked up in the 44−55 region accounting for 75% of all mutations detected by MLPA. Single exon deletions were found in 24 cases (36.4%) of which exon 45 was most commonly deleted (10 cases -15%), and exon 51 was deleted in 6 cases. Most common multiple exon deletions were at the 45-52 region in 7 cases (10.6%). Duplications accounted for 6.5% of all the mutations. Of the six duplications detected by MLPA, three were single exon duplications of exons 2, 45, and 52 [Table 1]. Longest duplication extended from exon 60 to 66, i.e., outside the hot spot region.
Muscle biopsy performed in the 11 MLPA-negative cases showed evidence of dystrophic features on routine histology in all these patients. IHC showed absent dystrophin staining in 4 (36.36%), sarcoglycan deficiency in 4 (36.36%), and in the remaining 3 (27.27%) cases, no deficiency was detected [Figure 3].
This is the first study from India and possibly in English literature to have compared the sensitivity of detection of mutations by mPCR and MLPA techniques in the same cohort of children with clinically and biochemically suspected cases of DMD and also to have looked at the detection rate by the gold standard method of IHC on muscle biopsy among the MLPA-negative cases.
By both methods, the deletions accounted for 79.5% (66/83) and duplications accounted for 7.22% (6/83), which differed from that observed among other Asian populations where duplication rates were considerably higher and to the extent of 27.3% in Korean  and 24.7% in Taiwanese patients,  as compared to deletions. Our findings were comparable to that seen those observed among among Caucasians where 63.4% deletions and 7.3% duplications were reported , and North Chinese populations, which showed 66.2% deletions and 6.25% duplications. 
Compared to mPCR positivity, the deletion detection rate by MLPA increased by 7.2% among our cohort as compared to 5.7% among other Asian populations. , The overall detection rate of our samples by MLPA was 86.8%, and thus, an increase by 14.5% was noted by combining both deletion and duplication rates, and this is similar to that observed among European patients where the improvement in detection rate was about 13%. 
Deletions and duplications are known to happen almost anywhere in the dystrophin gene. However, one location toward the central part of the gene (exons 44-55) and the other site toward the 5' end (exons 2-20) have been reported as the two hotspot regions. , In our study, the percentage of deletions restricted to the hotspot region decreased from 83.3% with mPCR to 75% with MLPA, suggesting that the overall high detection rate by MLPA could be attributed in part to the detection of additional mutations outside the hot spot region.
It is well known that the underlying mutation is not detectable in at least 4% of cases with available genetic testing, , due to a possible discrepancy between mutation study and the clinical phenotype. A muscle biopsy subjected for IHC and western blotting is indicated in patients without a detectable mutation. ,
Biopsy done for MLPA-negative patients in our study showed that 36.4% had absent staining for dystrophin antibody, thus confirming the diagnosis of DMD by the gold standard method in more than one-third of the MLPA-negative cases. However, we will not be able to offer genetic counseling without mutation analysis. Identification of Duchenne cases by IHC among the MLPA-negative patients is a clear indication of the possibility of detecting more unidentified mutations, and thus, if there are a high clinical suspicion and positive family history suggestive of DMD, subjecting the mutation-negative cases to novel methods like custom high-density comparative genomic hybridization array (CGH), which analyzes copy number variation across the entire dystrophin gene, and NGS of whole genomic DNA should be considered. Further, this is feasible as these tests are now available in India. ,, NGS has the added advantage of detecting complex rearrangements and large-scale intronic alterations, thus offering a higher mutation detection rate than MLPA and other exon-based tests.  However, it is apparent from the majority of genetic studies that all mutations cannot be identified with standard molecular analysis. In these small number of cases, a muscle biopsy may be helpful for protein studies and muscle RNA analysis to establish an accurate diagnosis.
Based on our observation in the suspected cohort of 83 DMD children, genetic study for the dystrophin gene analysis should begin with quantitative screening with MLPA testing, followed by full sequence analysis from genomic DNA. In the recent reports, MLPA-based array analysis system is a simple, rapid, and automated system, providing high resolution and speed. With the costs of advanced genetic tests becoming affordable, MLPA analysis should be enhanced by future technological improvements to further increase mutation detection and avoid the invasive muscle biopsy, which stops at a diagnosis and no further intervention.
The costs of MLPA and mPCR are comparable, and MLPA testing is even cheaper in certain laboratories. MLPA is a highly sensitive and rapid alternative to multiplex PCR. It can be used on blood samples, chorionic villi, and paraffin-embedded tissue. The ease of detection of duplications and the application for female carrier analysis are clearly the main advantages of the method. In our cohort, 36.36% of MLPA-negative cases still had DMD, and this emphasizes the need to consider more advanced genetic testing and consider performing a muscle biopsy only if it becomes mandatory for the diagnosis.
[Figure 1], [Figure 2], [Figure 3]